Project Summary/Abstract Clinical studies have demonstrated a significant loss of respiratory function with age. This results in reduced quality of life, increased propensity for other diseases, and ineffective aerosol drug delivery for the treatment of obstructive respiratory diseases. However, the cellular and molecular basis for age-dependent loss of respiration has not been established. Airway smooth muscle (ASM) plays a significant role in the regulation of respiration by influencing the bronchial tone due to its mechanical (contraction and relaxation) properties. Herein we propose to establish molecular changes that occur in the ASM due to aging that account for loss of respiratory function in the elderly. Preliminary studies demonstrate diminished contractile and relaxation responses of airways and ASM cells in aged (Old) rats compared to Young rats. Additional data suggest aging promotes phenotype plasticity or switching in ASM, a phenomenon observed in certain disease processes in which the smooth muscle contractile phenotype is changed to a proliferative/synthetic phenotype. Expression of myosin heavy chain and smooth muscle a-actin (contractile phenotype marker proteins) is lower in ASM obtained from Old rats compared to Young rats. Furthermore, global gene expression profile analysis in ASM cells revealed decreased expression of myostatin, a member of the TGF-b family, known to inhibit the growth and proliferation of myocytes. Lastly, preliminary data from ASM cells suggest diminished intracellular signaling with age to both contractile and relaxant agents that activate G protein-coupled receptors (GPCRs) on ASM. Based on these studies we hypothesize that ASM undergoes phenotype modulation with age that results in decreased contractile and relaxant responsiveness; a loss of GPCR signaling capacity underlies this loss of responsiveness, mediated in part by a loss of TGF-b pathway activation that occurs with age. In Specific Aim 1 we propose to establish age-dependent changes in ASM phenotype using rat and human airways and ASM cells by employing novel tools such as myograph, optical magnetic twisting cytometry and traction microscopy. In Specific Aim 2, we propose to establish the mechanistic basis for a phenotype switch in the ASM that occurs with age by detailing changes in the expression and activity of second messengers, effectors and regulators of GPCR signaling that regulate ASM function. In Specific Aim 3, we propose studies to characterize the effects of myostatin and TGF- b on airways and ASM phenotype, and determine the capacity of TGF- b signaling to reverse the synthetic/proliferative phenotype associated with age. Collectively, these studies seek to identify age-dependent molecular changes in ASM function that contribute to the age- associated decline in respiratory function in the elderly population. Our findings may help develop tools to improve respiratory functions, and modify diagnostic and treatment regimens for obstructive pulmonary diseases in the elderly.